The genus Ficus of the Moraceae family is a large genus including about 30 species. Ficus species are commonly used in gardening, in private as well as in public gardens. Most species bear fruit; however, in several species, fruit is considered a problem since the fruit are in-edible and falling fruit litter sidewalks and other pathways. The edible fruit, on the other hand, are highly desired as they are tasty and nutritional.
Fig trees are one of the earliest fruit bearing trees cultivated. Ficus carica L. (Moraceae), the well-known fig of commerce is indigenous to areas from Asiatic Turkey to North India, and natural varieties are cultivated in most of the Mediterranean countries. The fig is well known for its nutritive value, and is consumed fresh or as dried fruit worldwide. The fig fruits are also known for their mild laxative activity and high alkalinity, and substances derived therefrom are used in various drug preparations. Other parts of fig trees have also been shown to have a commercial value.
For example, U.S. Pat. No. 6,235,860 has recently disclosed the use of Ficus carica as a source for natural rubber.
U.S. Pat. No. 5,494,669 relates to a skin disorder known as pseudofolliculitis barbae (PFB), and more particularly to a preparation for treating PFB. The preparation disclosed is a topical solution comprising a mixture of alophatic alcohol, liquid aloe, liquid camphor, and the soluble materials of the fresh fig leaves of Ficus Carica. Similar compositions are disclosed as a massage composition (U.S. Pat. No. 4,582,706).
U.S. Pat. No. 6,184,193 discloses a shrinkage prevention agent for wet washing of clothing that would conventionally have been dry-cleaned. The shrinkage prevention agent disclosed comprises a steam or vacuum dry-distilled liquid of bark, leaves, stems or flowers of two or more plants, including Ficus carica. 
Traditional breeding methods of Ficus carica require a long-term effort for improving traits of fig trees. Among the Ficus carica some trees bear only female synconium and others, named caprifig, bear both male and female flowers. True fruits can be produced only on tress bearing female synconium and cross-pollination is mediated by a specific wasp. Thus, traits donated by the stamens are hard to track.
The application of genetic engineering techniques to stably incorporate homologous and/or heterologous genetic material into woody species, including fruit trees, offers the potential of obtaining improved planting stocks for private and public gardens and for agricultural use in a short period of time compared to those developed using traditional breeding techniques. In addition, efficient transformation methods can be used for the production of heterologous polypeptides having nutritional and/or pharmaceutical value, including edible vaccines.
Plants have the capacity to express foreign genes from a wide range of sources, including viral, bacterial, fungal, insect, animal, and other plant species. In single-copy nuclear transgenics, foreign protein in excess of 1% of total protein is often achieved (Hiatt et al., 1989. Nature 342:76-78). Further, assembly and processing of complex animal proteins in plants is possible, e.g., human serum albumin (Sijmons et al., 1990. Bio/Technology 8:217-220) and secretory antibodies (Ma et al., 1995. Science 268:716-719.). Expression of correctly processed avidin in corn seed at a level of 2% of the total soluble protein was also reported (Hood et al., 1997. Mol. Breeding. 3:291-306). It has been estimated that the cost of recombinant protein production in plants (assuming the foreign protein is 10% of total protein) can be 10 to 50 times less than in E. coli by fermentation (Kusnadi et al., 1997. Biotechnol. Bioeng. 56:473-484). Many plant species are now amenable to gene transfer, and numerous number of patents disclose different plant species capable of expressing foreign proteins; U.S. Pat. No. 6,392,121 discloses a system for gene amplification based on plant viral genetic elements that can be used to increase the production of foreign proteins within the plant.
Vaccines are administered to humans and animals to induce their immune systems to produce antibodies against viruses, bacteria, and other types of pathogenic organisms. In the economically advanced countries of the world, vaccines have brought many diseases under control. In particular, many viral diseases are now prevented due to the development of immunization programs. However, many vaccines for various diseases including poliomyelitis, measles, mumps, rabies, foot and mouth, and hepatitis B are still too expensive for the lesser-developed countries to provide to their large human and animal populations. Because of simplicity of delivery of vaccines by oral delivery, there is great current interest in discovering new oral vaccine technologies. Appropriately delivered oral immunogens can stimulate both humoral and cellular immunity and have the potential to provide cost-effective, safe vaccines for use in developing countries or inner cities where large-scale parenteral immunization is not practical or extremely difficult to implement. Such vaccines may be based upon bacterial or viral vector systems expressing protective epitopes from diverse pathogens (multivalent vaccines) or may be based upon purified antigens delivered singularly or in combination with relevant antigens or other pathogens. Several methods for using transgenic plants for oral immunization have been disclosed, for example International Patent Application WO 99/54452; U.S. Pat. Nos. 5,484,719; 5,612,487: 5,914,123; 6,034,320; 6,084,152; 6,194,123; 6,395,964 and 6,444,805.
The overall efficiency of techniques for genetically modifying plants depends upon the efficiency of the transformation technique(s) used to stably incorporate the required genetic material into plant cells or tissues, and the regeneration technique(s) used to produce viable plants from transformed cells.
Within Ficus species, there were some reports on regeneration and organogenesis from callus and explants. Ficus religiosa plants have been regenerated from callus of stem segments (Jaiswal and Narayan, 1985. Plant Cell Reports 4:256-258) and leaf callus (Narayan and Jaiswal, 1986. Ind. J. Exper. Bio1.24:193-194). Regeneration of Ficus lyrata plants from the axillary buds of a mature tree, and the formation of buds from leaf explants were also reported (Deshpande et al., 1998. Plant Cell Reports 17:571-573). However, for in vitro work with figs, plant regeneration has been restricted to the use of single shoot tips and apical buds.
Recently, Yakushiji et al. (J. of Hort. Sci. & Biotech. 2003, 78, 874-878) reported a method for the induction of organogenesis from leaf explants of Ficus carica using phloroglucinol (PG). However, by this method the frequency obtained for adventitious bud differentiation from leaf fragments was relatively low, and no adventitious buds were observed without PG. Moreover, regeneration was obtained only with non-transformed leaf segments. Although methods for transformation of woody plants have been reported (for example U.S. Pat. No. 6,255,559), no method for the introduction of isolated genetic material into Ficus species was hitherto disclosed.
Thus, there is a recognized need for, and it would be highly advantageous to have efficient methods for both transformation and regeneration of commercially valuable Ficus species, specifically fig trees, providing de novo origination of plant material from transformed cells and development of the genetically modified plant material to produce genetically modified plants.